Charomids are cosmid vectors up to 52 kilobases (kb) long, bearing 1-23 copies of a 2-kb spacer fragment linked in head-to-tail tandem arrays. Like cosmids and lambda phage, charomids can be packaged in vitro for efficient introduction into bacteria. Charomids contain a polylinker with nine unique restriction sites for cloning and can be used without preparing vector arms. Using a charomid of appropriate size, one can clone inserts of any size up to 45 kb. For example, charomid 9-36 (9 cloning sites, 36 kb long) is too small to be packaged efficiently without an insert and can be used to clone fragments of 2-16 kb. The structure of charomids facilitates restriction mapping of the insert DNA and, after cloning, all the spacer fragments can be removed easily. After enrichment by size fractionation in an agarose gel, a specific single-copy genomic sequence can be cloned rapidly from approximately 3 micrograms of DNA. Using charomid 9-36, we have cloned and mapped an amplified novel DNA fragment from a cell line resistant to N-(phosphonoacetyl)-L-aspartate and carrying about 100 copies of the CAD (carbamoyl-phosphate synthetase/aspartate carbamoyltransferase/dihydroorotase) gene. The fragment lies at the center of an inverted duplication of this gene.